Crystallization of proteins is an important requirement in determining protein structure. X-ray crystallography or linear accelerator (cyclotron) characterization techniques are currently used in determining protein structure. Protein crystallography is used to ascertain the three-dimensional molecular structure of protein crystals. This is essential for understanding the biological functions attributed to these macromolecules. The physical shape and folding of a protein is of increasing importance to drug companies interested in rational drug design. Drug molecules are designed to fit exactly into a binding site of a macromolecule, thus blocking its function in a given disease pathway. Producing higher quality crystals results in more accurate modelling of the 3-dimensional protein structures and consequently more efficacious drugs. This accuracy is referred to as the resolution of the structure. The larger and more perfect crystals provide the highest resolution.
Perfect crystals are difficult to achieve on Earth. Ambient gravity and turbulence disrupt crystal formation in that terrestrial samples mix as a result of gravity-driven convective flow. Therefore a microgravity environment promotes better crystal formation, in part due to the lack of turbulence and mixing within a liquid or gaseous sample during crystal formation. Spacecraft in low Earth orbits can provide a microgravity environment that is convection- and sedimentation-free for the study and applications of fluid-based systems. With the advent of the Space Shuttle, scientists had regular access to such environments and many experiments were initiated, including those in protein crystallization. After many trials it became clear that for several proteins, crystallization in a microgravity environment resulted in bigger and better quality crystals. The generation of perfect crystals can sometimes be the limiting factor in determining a protein's structure. By eliminating variables such as gravity, crystals are able to form slower and more precise in space.
Given the great expense required for crystallization studies in space, prior art methodologies to form crystals have largely been terrestrial. The leading technique is the “hanging drop” technique in which a protein in a liquid solution is allowed to “hang” from a well within a specially designed tray, and the liquid gradually evaporates, leaving only the protein crystal. However, the quality of crystals can be compromised in the absence of a microgravity environment. Also, the hanging drop method requires transfer of a crystal from a well within a tray into a micropipette. Further, removal of the vapor to enhance crystal growth is not common in the prior art. Gases (such as argon) are heated and blown over the protein sample of interest; the use of these gases is cumbersome and can lead to contamination. Furthermore, the use of gases renders it difficult to control the reproducibility of the method.
U.S. Pat. No. 6,458,332 issued to Ooshima et al., discloses a device for forming protein crystals from solution. The device is a tank with a temperature differential between the top and the bottom of the tank. A protein-containing fluid is placed in the tank, and the temperature differential forces a portion of the fluid into channels submerged within the fluid and leading upward toward the top of the tank. When the fluid channels are rotated about a central vertical axis, the fluid is sprayed against the top wall of the tank, which causes evaporation and crystal formation. This device does not appear to be suited to a microgravity environment, as gravity appears to be required to maintain the fluid at the bottom of the tank.
U.S. Pat. No. 6,387,399 issued to Morrison et al., describes a method by which crystals are formed under microgravity conditions by encapsulating the protein and exposing it to an osmotically pressurized environment to effect de-watering. Other related U.S. patents include: U.S. Pat. Nos. 6,214,300; 6,103,271; 6,099,864; 6,015,104; and 5,827,531.
The sitting drop vapor diffusion technique is another method for the crystallization of macromolecules. Based on the same principles, a drop of a mixture of sample and reagent is placed in vapor equilibration with a liquid reservoir of reagent. Typically, the drop contains a lower reagent concentration than the reservoir. To achieve equilibrium, water vapor leaves the drop and eventually ends up in the reservoir. As water leaves the drop, the sample undergoes an increase in relative supersaturation. Both the sample and reagent increase in concentration as water leaves the drop for the reservoir. Equilibration is reached when the reagent concentration in the drop is approximately the same as that in the reservoir. One disadvantage of this is the adherence of crystals to the sitting drop surface making removal of the crystals difficult for further analysis. A disadvantage of both sitting drop and hanging drop techniques is that the initial vaporization (before nucleation of the seed crystal) is favored by a slow process. Increased or uncontrolled rate of vaporization can diminish the quality of the crystals.
Temperature can be a significant variable in biological macromolecule and small molecule crystallization. Temperature often influences nucleation and crystal growth by manipulating the solubility and supersaturation of the sample. Thus the control of temperature during crystal production is essential for successful and reproducible crystal growth of proteins with temperature dependent solubility. An advantage is that a temperature gradient provides precise, quick, and reversible control of relative supersaturation. Using temperature in addition to standard crystallization variables (such as sample concentration, reagent composition and concentration, and pH), can increase the probability of producing crystals as well as uncover new crystallization conditions for a sample. Protein solution temperature can be used to carefully manipulate crystal nucleation and growth. This control can also be used to etch or partially dissolve then grow back the crystal in an attempt to improve crystal size, morphology, and quality. Temperature control is noninvasive and can manipulate sample solubility and crystallization with altering reagent formulation.
While controlled temperature can be important for consistent results, temperature fluctuation of the protein solution can be useful in obtaining high quality crystals since for a sample with temperature dependent solubility, changes in temperature can equate to changes in a crystallization reagent condition. Temperature gradients can be used to optimize the use of proteins with temperature-dependent solubility. To achieve this, the experiment is equilibrated at one temperature then slowly changed to a second temperature. The above approach may be useful but does not support a reproducible generic process applicable to each protein.
U.S. Pat. No. 6,406,903 issued to Bray et al., teaches a protein crystallization system employing temperature-based precipitation of proteins from solution, and uses humidity or temperature detectors to dynamically control the conditions within the system. A vapour diffusion system is described in which a protein-containing solution is exposed to a precipitant solution, causing water vapor to diffuse away from the protein solution. Also, a temperature-based system is described in which the temperature of a protein-containing solution is raised or lowered to initiate or promote crystal growth. It may employ a water-cooled heatsink. Although temperature fluctuations are used, this patent does not disclose the maintenance of a temperature differential.
U.S. Pat. No. 4,886,646 issued to Carter et al., relates to the aforementioned “hanging drop” method of crystal growth in which a drop of an aqueous protein-containing solution is allowed to hang over a well and evaporate to form a crystal. A flow of control fluid near the hanging drop is used to create a vapour pressure gradient and to withdraw water from the drop. However, it does not use a temperature modification method to create a temperature gradient. The hanging drop method and the effect of temperature changes on condensation, nucleation and crystal growth are discussed in a technical paper by Hampton Research Corporation (2001).
U.S. Pat. No. 4,755,363 issued to Fujita et al., describes a system for forming protein crystals using a variety of solvents. It can be adapted to different methods of forming a crystal, including a batch-wise method, a vapour diffusion method, and a free interface diffusion method, each of which employs concentrated solvents to draw water vapour out of a protein-containing solution. The system described includes a temperature control unit, and the role of changing temperature gradually within a closed system is discussed as a way of controlling crystal growth.
U.S. Pat. No. 6,409,832 issued to Weigl et al., discloses a device for promoting protein crystallization from solution. It employs a solvent-based laminar flow methodology, in which a protein is crystallized by exposure to a solvent within a microfluidic channel, effecting de-watering of the protein.
U.S. Pat. No. 5,643,540 issued to Carter et al., discloses a closed system for forming protein crystals in microgravity. The system uses concentrated solvents to form a vapour pressure differential with a protein-containing solution, but not a temperature differential. Further, the level of vaporation is not controlled.
It is, therefore, desirable to provide a temperature- and vapor-controlled protein crystallization device that can be used under conditions of microgravity.